Immunoassay test slide

ABSTRACT

An immunoassay test slide for use in a dry chemistry analytical instrument includes a slide housing or case formed from two matable sections—a slide cover piece and a slide bottom piece. The slide housing defines an interior cavity in which is situated a sheet-like porous carrier matrix. The slide cover piece has an opening formed through the thickness thereof to expose a central portion of the fluid flow matrix so that a precise volume of fluid sample of blood, serum or the like, preferably pre-mixed with a conjugate reagent, and precise volumes of a wash reagent and a substrate (detector reagent), may be deposited on the matrix through the cover opening by a metering device of the analytical instrument. The bottom piece of the immunoassay test slide is transparent, and the slide is moved by a transport mechanism of the analytical instrument over a reflectometer or a fluorometer for performing reflectance or fluorescence measurements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/358,280, filed on May 15, 2014, and entitled “Immunoassay TestSlide”, which claims the benefit of priority, under 35 U.S.C. 371, tointernational PCT Application No. PCT/US2013/020483, filed on Jan. 7,2013, and, under 35 U.S.C. 119 and/or 35 U.S.C. 120, to U.S. ProvisionalApplication Ser. No. 61/585,050, filed on Jan. 10, 2012, and entitled“Immunoassay Test Slide”, the disclosure of each of which isincorporated herein by reference and on which priority is herebyclaimed.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to devices and methods for performingassays to determine the presence or quantity of a specific analyte ofinterest in a fluid sample.

Description of the Prior Art

Many devices for the detection and/or quantification of an analyte ofinterest in a fluid sample are well known and are the lateral-flow typeor micro-well type. Generally, lateral flow devices include a solidphase fluid permeable flow path through which a sample and variousreagents travel by capillary action. The flow path has immobilizedthereon various binding reagents for the analyte (or analog thereof),other binding partners, or conjugates involving binding partners for theanalyte and members of signal producing systems (e.g., a label). Thevarious assay formats used with these devices are well known for thedirect or indirect detection of the analyte of interest in the testsample.

U.S. Pat. Nos. 5,726,010, 5,726,013, 5,750,333 and 7,816,122, each ofwhich issued to Scott M. Clark and which are assigned of record to IDEXXLaboratories, Inc., the disclosures of which are incorporated herein byreference, describe assay methods and devices that use the formation ofa solid phase bound tertiary complex to detect an analyte of interest ina fluid sample. The devices disclosed in the Clark patents utilize areversible flow in a chromatographic binding assay. Ananalyte-containing solution is applied to the device and then istransported by capillary action, first in one direction and then in theopposite direction, along an elongated flow matrix. The flow matrixgenerally includes four different regions. Region one is where asolution having the sample mixed with either a labeled antibody orantigen is added. Region two, also called the detection zone, containsthe second antibody or antigen, which is immobilized to a solid phase.Region three contains a site to apply a wash solution. Region fourcontains an absorbent reservoir located near region one and makes theflow go in the opposite direction. The device also includes means todetect the presence or quantity of an analyte.

The reversible lateral flow device disclosed in the Clark patents worksquite well in detecting an analyte in a fluid sample. However, it, likeother lateral flow devices, requires relatively significant samplevolume and other reagents so that the matrix can be sufficiently wettedto allow for lateral flow of the sample liquid, wash and substrate. Morespecifically, such lateral flow devices may require approximately 0.35grams (0.35 milliliters) of the sample liquid. Thus, samples often needto be diluted when sample volumes are small.

Dry chemical reagent test slides having a film surrounded by a frame arealso well known in the art and are used to analyze a blood or fluidsample deposited thereon in a chemical analyzer, such as disclosed inU.S. Pat. No. 5,089,229 (Heidt, et al.) and U.S. Patent ApplicationPublication No. 2010/0254854 (Rich, et al.), the disclosures of whichare incorporated herein by reference. The sample deposited on the slidesreacts with the chemical reagent on the film, and the reflectance orfluorescence of the slides is then measured by the chemical analyzer todetect a compound or substance found in the sample, such as calcium(Ca), aspartate transminase (AST) or glucose (Glu), which could be anindication of a condition or disease. Only small aliquots of samplefluid need be deposited on the slides for detection of certainindicators of diseases. It would be advantageous if immunoassays couldbe performed on such dry chemistry analytical instruments using testslides.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide an immunoassay testslide formed in accordance with the present invention which may be usedin a dry chemistry analytical instrument.

It is another object of the present invention to provide a method inaccordance with the present invention for manufacturing such animmunoassay slide.

It is a further object of the present invention to provide a method inaccordance with the present invention for performing assays in a drychemistry analytical instrument using the immunoassay test slide of thepresent invention.

In accordance with one form of the present invention, an immunoassaytest slide for use in a dry chemistry analytical instrument, and forperforming assays for detecting the presence or quantity of an analyte(e.g., an antigen or antibody, and the like), includes a slide housingor case formed from two matable sections—a generally planar slide coverpiece and a generally planar slide bottom piece joinable to the coverpiece. The slide housing formed from the cover piece and bottom piece,when joined together, is substantially leakproof during use and definesan interior cavity. A sheet-like porous carrier matrix is disposedwithin the confines of the housing cavity.

The slide cover piece has an opening formed through the thicknessthereof to expose a central portion of the fluid flow matrix. Theopening in the cover piece is provided to expose a central portion ofthe porous carrier matrix so that a precise volume of fluid sample(e.g., blood, serum and the like), preferably pre-mixed with a conjugatereagent, as will be explained, a wash reagent and a substrate (detectorreagent), may be deposited on the matrix through the cover opening by ametering device of the analytical instrument. The central portion of thecarrier matrix has deposited thereon a dried and immobilized specificbinding reagent situated in alignment with the central opening in thecover piece.

The bottom piece of the immunoassay test slide also includes a centralopening formed through the thickness thereof, which opening may becovered by a thin sheet of transparent (clear) material, such as Mylar,to avoid contamination and maintain the leakproofness of the housing.The opening in the bottom piece is provided so that reflectance orfluorescence measurements may be made of the immunoassay slide as it istransported by the analytical instrument over a reflectometer orfluorometer forming part of the analytical instrument. Alternatively,the bottom piece of the immunoassay test slide may be formed of atransparent material, in lieu of having the opening formed in the bottompiece, in order to conduct such measurements on the slide.

The overall shape of immunoassay test slide is preferably eitherrectangular or square, similar to the chemical reagent test slidesdisclosed in the aforementioned Heidt, et al. '229 patent (U.S. Pat. No.5,089,229), or trapezoidal, similar to the chemical reagent test slidesdisclosed in the aforementioned Rich, et al. published application(Publication No. 2010/0254854). The thickness of the immunoassay testslide is preferably the same as or slightly greater than that ofconventional dry chemistry slides so that they may be useable withexisting dry chemistry analytical instruments which accept such drychemistry slides, as disclosed in the aforementioned Heidt, et al.patent and Rich, et al. published application.

In accordance with a method of using the immunoassay test slide on a drychemistry analytical instrument to perform an assay, the immunoassaytest slide is loaded into a transport mechanism of the analyticalinstrument, which moves the slide under a sample metering device andabove a reflectometer or fluorometer. An aliquot of fluid samplecontained in a vial is also loaded into the analytical instrument. Thefluid sample may have been pre-mixed with a conjugate reagent prior tobeing loaded into the analytical instrument, or mixed with the conjugatereagent by the instrument. Depending upon the assay format, theconjugate reagent may specifically bind to an analyte in the fluidsample to form a complex of the analyte and the conjugate reagent. Inanother aspect, the conjugate reagent includes an analyte analog, whichdoes not complex with the analyte. The sample/conjugate reagent mixtureis then incubated for a predetermined period of time.

Then, a predetermined volume of sample/conjugate is metered onto theimmunoassay test slide through the opening formed in the cover piece bythe metering device of the analytical instrument. The sample liquidcontaining the analyte and the conjugate reagent, whether complexed ornot, flows into the central portion of the matrix located at the topopening in the cover piece and is transported by capillary action in alldirections within the matrix. After that, a series of washes of the testslide is performed by having the metering device of the analyticalinstrument deposit predetermined volumes of a wash reagent on to theslide through the top opening of the cover piece. Finally, apredetermined volume of a substrate, such as a detector reagent, isadded to the slide through the top opening by the metering device of theanalytical instrument.

Reflectance or fluorescence measurements are then taken of the slidethrough the clear bottom piece or bottom opening of the slide at aparticular wavelength. The presence or quantity of a specific analyte ofinterest in the fluid sample may be determined by such measurements.Preferably, there is a detectable color reaction on the slide which ismeasured by the analytical instrument that is used in the detection andquantification of the analyte in the fluid sample.

The immunoassay test slide of the present invention may be formed byplacing a die cut section of porous carrier matrix from a sheet of thesame material between a cover piece and a bottom piece of a plasticmaterial, such as polystyrene, specifically shaped to be matable. Thetwo pieces may be joined together by applying heat or an adhesive todefine a substantially leakproof housing in which resides the porouscarrier matrix. The porous carrier matrix may be spotted with animmobilized specific binding reagent prior to its insertion between thetwo mating slide pieces, or may be spotted with the specific bindingreagent and heated to a specific temperature and for a predeterminedperiod time to dry and immobilize the binding reagent in the centralportion of the matrix under the opening in the cover piece. If a bottomopening, formed in the bottom piece of the immunoassay test slide, isprovided, then prior to the insertion of the porous carrier matrixbetween the cover piece and the bottom piece, a thin sheet oftransparent (clear) material, such as Mylar, is placed within theinterior cavity defined by the slide housing over the bottom opening.Alternatively, no such bottom opening or covering sheet is required ifthe bottom piece of the slide is formed from a light transmissible ortransparent material.

These and other objects, features and advantages of the presentinvention will be apparent from the following detailed description ofillustrative embodiments thereof, which is to be read in connection withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded, top perspective view of one form of animmunoassay test slide constructed in accordance with the presentinvention.

FIG. 2 is an exploded, side elevational view of the immunoassay testslide of the present invention shown in FIG. 1.

FIG. 3 is a top plan view of the immunoassay test slide of the presentinvention shown in FIG. 1.

FIG. 4 is an exploded, top perspective view of yet another embodiment ofthe immunoassay test slide formed in accordance with the presentinvention.

FIG. 5 is a graph of optical reflectance versus time showing theprogress curve for a two-step test protocol using an fPL immunoassaytest slide formed in accordance with the present invention.

FIG. 6 is a graph of optical reflectance versus nanograms/millilitersshowing the calibrator dose curve for a two-step test protocol using anfPL immunoassay test slide formed in accordance with the presentinvention.

FIG. 7 is a graph of optical reflectance versus nanograms/millilitersshowing the calibrator dose curve for a one-step test protocol (singlepremix dispense) using an fPL immunoassay test slide formed inaccordance with the present invention.

FIG. 8 is a graph of optical reflectance versus nanograms/millilitersshowing the calibrator dose curve for a one-step test protocol (multiplepremix dispense) using an fPL immunoassay test slide formed inaccordance with the present invention.

FIG. 9 is a top perspective view of a further embodiment of animmunoassay test slide formed in accordance with the present invention.

FIG. 10 is a bottom perspective view of the immunoassay test slide ofthe present invention shown in FIG. 9.

FIG. 11 is an exploded, top perspective view of the immunoassay testslide of the present invention shown in FIGS. 9 and 10.

FIG. 12 is an exploded, bottom perspective view of the immunoassay testslide of the present invention shown in FIGS. 9-11.

FIG. 12A is a top perspective view of the immunoassay test slide of thepresent invention having an alternative shape than that shown in FIGS.9-12.

FIG. 13 is a plan view of the outer side of a cover piece of animmunoassay test slide formed in accordance with the present invention.

FIG. 14 is a plan view of the inner side of the cover piece of theimmunoassay test slide formed in accordance with the present invention.

FIG. 15 is a side view of the cover piece of the immunoassay test slideof formed in accordance with the present invention.

FIG. 16 is a cross-sectional view of the cover piece of the immunoassaytest slide of the present invention taken along line 16-16 of FIG. 14.

FIG. 17 is a plan view of the outer side of a bottom piece of animmunoassay test slide formed in accordance with the present invention.

FIG. 18 is a plan view of an inner side of the bottom piece of theimmunoassay test slide formed in accordance with the present invention.

FIG. 19 is a side view of the bottom piece of the immunoassay test slideformed in accordance with the present invention.

FIG. 20 is a cross-sectional view of the bottom piece of the immunoassaytest slide of the present invention taken along line 20-20 of FIG. 18.

FIG. 21A is diagrammatic illustration of a method of using theimmunoassay test slide of the present invention to detect an analyte ina sample fluid.

FIG. 21B is a graph of reflectance measurements versus time illustratingexemplary results of tests performed on a T4 slide in accordance withthe method of the present invention shown in FIG. 21A.

FIG. 22 is a front isometric view of a chemical analyzer formed inaccordance with one form of the present invention for use with bothconventional dry chemistry analytical slides and the immunoassay testslide of the present invention.

FIG. 23 is a front isometric view of the chemical analyzer shown in FIG.22, illustrating a first sliding door on the front face of the analyzerbeing open and a first slide inserter mechanism of the chemical analyzerextending beyond the front face of the analyzer housing.

FIG. 24 is a front isometric view of the chemical analyzer, with thehousing thereof partially broken away, and illustrating variouscomponents of the analyzer of the present invention.

FIG. 25 is a top isometric view of a reflectometer used in the chemicalanalyzer of the present invention, with portions of the reflectometerpartially broken away.

FIG. 26 is a cut-away pictorial illustration of a fluorometer used inthe chemical analyzer of the present invention.

FIG. 27 is a front isometric view of a portion of the slide insertermechanism used in the chemical analyzer of the present invention, andillustrating the placement of either a centrifuge rotor or a sample vialthereon.

FIG. 28 is a front isometric view of the chemical analyzer of thepresent invention shown in FIG. 22, and illustrating the extension fromthe front face of the analyzer housing of a tray for carrying a diluentand mixing cup or other cups for containing reagents and the like.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring initially to FIGS. 9-20 of the drawings, it will be seen thatan immunoassay test slide 2 constructed in accordance with the presentinvention includes a substantially leakproof housing or case 4 formed oftwo sections—a cover piece 6, and a bottom piece 8 joinable to the coverpiece 6. The housing or case 4 defined by the cover piece 6 and thebottom piece 8, when joined together, defines an interior cavity 10 inwhich is situated an absorbent material 12, also referred to herein as aporous carrier matrix or a fluid flow matrix, as will be described ingreater detail.

The cover piece 6 and the bottom piece 8 of the housing 4 are formedfrom a plastic material, such as polystyrene, polypropylene orpolyethylene. Each of the cover piece 6 and the bottom piece 8 includesan inner surface 14, 18, facing the interior cavity 10 of the housing 4,and an opposite outer surface 22, 24 which is exposed. The bottom piece8 includes one or more projections or ribs 28 extending outwardly fromthe inner surface 18 of the bottom piece, which ribs 28 are set slightlyinwardly from the peripheral edge of the bottom piece 8. The ribs 28 areprovided to help secure the cover piece 6 to the bottom piece 8, i.e.ribs 28 provide material that can flow during a welding operation.

The cover piece 6 includes a continuous side wall 30 which extends aboutthe periphery of the cover piece. The preferred thickness of the sidewall 30 of the cover piece 6 is preferably equal to the distance theribs 28 of the bottom piece 8 are set inwardly from the peripheral edgethereof so that, when the cover piece 6 is mounted on the bottom piece8, the lower edge of the side wall 30 of the cover piece rests on theinner surface 18 of the bottom piece 8, with the outer surface of theside wall 30 being flush with the peripheral edge of the bottom piece 8,and further with the ribs 28 of the bottom piece being situated incontact with or in close proximity to the inner surface of the side wall30 of the cover piece. Of course, it should be realized that thestructure described previously with respect to the cover piece 6 and thebottom piece 8 may be reversed, that is, with the ribs 28 situated onthe cover piece 6, and the side wall 30 situated on the bottom piece 8,and such structure is envisioned to be within the scope of the presentinvention.

The cover piece 6 and the bottom piece 8 are joined together with anadhesive or by heat sealing the two pieces together so as to form asubstantially leakproof seal for the housing 4 of the immunoassay testslide. Preferably, portions of the cover piece 6 and bottom piece 8 maybe joined together by melting those portions, such as by sonic weldingor heat stamping, those portions being allowed to harden and fusetogether.

For added strength, and to further facilitate the positioning of thecover piece 6 with respect to the bottom piece 8 when the two pieces arejoined together, the cover piece 6 may include one or more (preferablyfour) posts 32 which extend perpendicularly outwardly from the innersurface 14 of the cover piece 6 a predetermined distance into theinterior cavity 10. In addition, the bottom piece 8 may include one ormore (preferably four) columns or supports 34 extending perpendicularlyoutwardly from the inner surface 18 of the bottom piece 8 apredetermined distance into the interior cavity 10. Each column orsupport 34 of the bottom piece includes a bore 36 formed at leastpartially axially therein, which is dimensioned to receive acorresponding post 32 of the cover piece 6. The columns or supports 34of the bottom piece 8 are positioned to be in alignment with the posts32 of the cover piece 6 so that, when the cover piece 6 is mated to thebottom piece 8, the posts 32 of the cover piece are received by thebores 36 in the supports or columns 34 of the bottom piece, with thefree ends of the posts 32 preferably resting on the inner surface 18 ofthe bottom piece 8 within their respective columns or supports 34. Theposts 32 and columns 34 respectively of the cover piece 6 and bottompiece 8 add further strength and rigidity to the immunoassay test slide2, especially for the interior portion thereof, and help maintain theoverall thickness of the housing 4 of the immunoassay test slide 2 to adesired dimension. Of course, it should be realized that the positioningof the posts 32 and columns 34 may be reversed, that is, with the posts32 extending outwardly from the inner surface 18 of the bottom piece 8,and the columns 34 extending outwardly from the inner surface 14 of thecover piece 6, and such structure is envisioned to be within the scopeof the present invention.

The cover piece 6 of the immunoassay test slide 2 has an opening 38formed through the thickness thereof which may be circular, rectangularor, as shown in the drawings, oval in shape. This top opening 38 isprovided so that a precise amount of a sample fluid, such as blood,serum and the like, and reagents may be metered onto the test slide 2and deposited on the absorbent material 12 (the porous carrier matrix orfluid flow matrix) situated under the opening 38, by a sample meteringdevice of the dry chemistry analytical instrument, such as disclosed inthe aforementioned Heidt, et al. '229 patent and the Rich, et al.published application, as will be described in greater detail.Furthermore, the bottom piece 8 may have an opening 40 formed throughthe thickness thereof, which bottom opening 40 is situated in alignmentwith the top opening 38 of the cover piece 6 when the two pieces aremated together. The bottom opening 40 is provided so that light emittedby a reflectometer or fluorometer of the analytical instrument may passtherethrough and impinge on the fluid flow matrix 12 within theimmunoassay test slide 2, and be reflected or fluoresced thereby, thereflected or fluoresced light being detected by the reflectometer orfluorometer during measurements conducted on the immunoassay test slideas it is moved over the reflectometer or fluorometer by a transportmechanism of the analytical instrument. The bottom opening 40, like thetop opening 38, may be rectangular, oval or, as shown in the drawings,circular in shape.

Both the top opening 38 in the cover piece 6 and the bottom opening 40in the bottom piece 8 are situated substantially centrally on eachrespective piece and in alignment with each other, and are furthersituated essentially between the four posts 32 and columns 34.

If a bottom opening 40 provided, then a transparent or clear (lighttransmissive) thin sheet of material 42, such as Mylar, may be placedover the bottom opening and adhesively joined or heat sealed to theinner surface 18 the bottom piece 8 within the interior cavity 10 of thehousing, that is, interposed between the inner surface 18 of the bottompiece and the absorbent porous material 12, to insure the leakproofnessof the housing 4. Alternatively, the bottom piece 8 may be formed from atransparent or clear (light transmissive) material (glass or plastic,for example), and the bottom opening may be omitted. More specifically,with respect to this alternative embodiment, the material from which thebottom piece 8 is fabricated is chosen to allow visible or infraredlight, or more preferably, light at a wavelength of about 645nanometers, to permeate therethrough. Light emitted by the reflectometeror fluorometer of the analytical instrument will pass through thetransparent Mylar cover 42 or the transparent bottom piece 8 to impingeon the absorbent material 12 when the analytical instrument isconducting reflectance or fluorescence measurements on the immunoassaytest slide 2.

The housing 4 of the immunoassay test slide defined by the cover piece 6and the bottom piece 8, when joined together, preferably has an overallrectangular shape like that of the conventional dry chemistry reagenttests slides disclosed in the aforementioned Heidt, et al. '229 patent,or an overall trapezoidal shape like the chemical reagent test slidesdisclosed in the aforementioned Rich, et al. published application.Thus, the housing 4 includes a front wall 44, a rear wall 46 situatedopposite the front wall, and two opposite lateral walls 48, each ofwhich is defined at least in part by the side wall 30 of the cover piece6. If the immunoassay test slide housing 4 takes on a rectangular shape,then each wall 44-48 is perpendicularly joined to its next adjacentwall, as shown in FIG. 12A. If the immunoassay test slide housing has atrapezoidal shape, such as shown in FIGS. 9-12 and 12A-20, then thefront and rear walls 44, 46 are generally parallel to each other and therear wall 46 has a length which is greater than that of the front wall44, and the opposite lateral walls 48 are non-parallel to each other andmutually converge from the rear wall 46 toward the front wall 44.

Furthermore, the overall dimensions of the immunoassay test slide 2, andthe overall thickness thereof, are substantially the same as thedimensions and thickness of the dry chemistry reagent tests slidesdisclosed in the Heidt, et al. patent and the Rich, et al. publishedapplication. In this way, the immunoassay test slides 2 may be used withsuch analytical instruments disclosed in the Heidt, et al. patent andthe Rich, et al. published application, and with other conventionalanalytical instruments, just like the dry chemistry test slides having aframe surrounding a film portion carrying a reagent, as disclosed in theaforementioned Heidt, et al. patent and the Rich, et al. publishedapplication.

In addition, each of the cover piece 6 and the bottom piece 8 is formedto include, and to define the housing 4 of the immunoassay test slide 2with, an indexing notch 50 for proper orientation of the test slide onthe analytical instrument, and lateral side recesses 54 used for loadingthe test slides on the analytical instrument, in the same manner and inthe same locations as the notch and lateral side recesses included inthe dry chemistry test slides disclosed in the aforementioned Heidt, etal. patent and the Rich, et al. published application. It should benoted that the side wall 30 of the cover piece 6 extends about the notch50 and the lateral side recesses 54 to insure that the housing 4 issubstantially leakproof.

As mentioned previously, an absorbent material 12 (i.e., the porouscarrier matrix or fluid flow matrix) is disposed within the confines ofthe housing cavity 10. The fluid flow matrix 12 is preferably die cutfrom a sheet of such material and shaped to conform to the innerdimensions of the housing 4. More specifically, and as can be seen inthe drawings, the fluid flow matrix 12 includes a notched-out portion 56and recessed side portions 58 to accommodate the notch 50 and lateralrecesses 54 formed in the housing 4, and includes four cutouts 60 formedthrough its thickness which are aligned with and dimensioned to receivethe columns or supports 34 of the bottom piece 8 so as not to interferewith the ability of the posts 32 of the cover piece 6 being received bythe bores 36 of the columns of the bottom piece 8. Thus, the columns 34and posts 32 respectively of the bottom piece 8 and cover piece 6,passing through the thickness of the fluid flow matrix 12, help to holdthe fluid flow matrix in place within the interior cavity 10 of thehousing, without shifting.

As further mentioned previously, the fluid flow matrix 12 is dimensionedand shaped to fit within the confines of the interior cavity 10 of thetest slide housing 4. Preferably, however, the matrix is dimensioned tobe slightly smaller than the dimensions of the interior cavity 10 sothat its lateral edges are spaced slightly away from the inner surfaceof the side wall 30 of the cover piece 6 to define a channel or well 62(see FIG. 20) between the matrix 12 and side wall 30 at least partiallyabout the periphery of the housing. This channel or well 62 is providedto receive any overflow of fluid sample, reagent or wash solution fromthe matrix 12 which is envisioned to become saturated with such fluids.The well or channel 62 provides capacity in excess of the volume of thefluid sample, reagents and wash solutions saturating the fluid flowmatrix 12. The preferred volume of the interior cavity 10 defined by thehousing 4 of the immunoassay test slide is between about 20 microlitersand about 200 microliters, and is preferably about 270 microliters. Invarious alternative embodiments of the immunoassay test slide 2 of thepresent invention, the absorbent matrix 12 may have a volume thatoccupies about 50 percent, or about 60 percent, or about 70 percent, orabout 80 percent, or about 90 percent, of the interior space 10 definedby the housing.

The flow matrix material preferably possesses the followingcharacteristics: (1) low non-specific affinity for sample materials andlabeled specific binding reagents, (2) ability to transport a liquid bycapillary action over a distance with a consistent liquid flow acrossthe matrix 12, and (3) ready binding to immobilized specific bindingreagents (e.g., by covalent or non-covalent attachment or by physicalentrapment). Materials possessing these characteristics include fibrousmats composed of synthetic or natural fibers (e.g., glass orcellulose-based materials or thermoplastic polymers, such as,polyethylene, polypropylene, or polyester); sintered structures composedof particulate materials (e.g., glass or various thermoplasticpolymers); or cast membrane films composed of nitrocellulose, nylon,polysulfone or the like (generally synthetic in nature). The inventionmay utilize a flow matrix 12 composed of sintered, fine particles ofpolyethylene, commonly known as porous polyethylene, such as sinteredpolyethylene beads; preferably, such materials possess a density ofbetween 0.35 and 0.55 grams per cubic centimeter, a pore size of between5 and 40 microns, and a void volume of between 40 and 60 percent.Particulate polyethylene composed of cross-linked or ultra highmolecular weight polyethylene is preferable. A flow matrix 12 composedof porous polyethylene possesses all of the desirable features listedabove, and in addition, is easily fabricated into various sizes andshapes. A particularly preferred material is 10-15 micron porouspolyethylene from Chromex Corporation FN #38-244-1 (Brooklyn, N.Y.).Another preferred material is Fusion 5™ lateral flow matrix availablefrom Whatman, Inc., USA.

The porous carrier matrix 12 may be made from a material which has a lowaffinity for the analyte and test reagents. This is to minimize or avoidpretreatment of the test matrix to prevent nonspecific binding ofanalyte and/or reagents. However, materials that require pretreatmentmay provide advantages over materials that do no require pretreatment.Therefore, materials need not be avoided simply because they requirepretreatment. Hydrophilic matrices generally decrease the amount ofnon-specific binding to the matrix 12.

In one aspect, the porous carrier matrix 12 may have an open porestructure with an average pore diameter of 1 to 250 micrometers and, infurther aspects, about 3 to 100 micrometers, or about 10 to about 50micrometers. The matrices 12 are from a few mils (0.001 in) to severalmils in thickness, typically in the range of from about 10 mils to about20 mils and, most preferably, about 16 mils.

An example of a suitable porous carrier matrix 12 in whichomni-directional flow occurs is the high density or ultra high molecularweight polyethylene sheet material manufactured by Porex TechnologiesCorp. of Fairburn, Ga., USA. This material is made from fusing sphericalparticles of ultra-high molecular weight polyethylene (UHWM-PE) bysintering. This creates a porous structure with an average pore size ofeight to 20 microns, depending on the size of the particles (20 to 60microns, respectively). The polyethylene surface is treated with anoxygen plasma and then coated with alternating layers ofpolyethylenimine (PEI) and poly acylic acid (PAA) to createsurfactant-free hydrophilic surface having a wicking rate of 0.01-0.5cm/s.

While matrices 12 made of polyethylene have been found to be highlysatisfactory, omni-directional flow materials formed of other olefin orother thermoplastic materials, e.g., polyvinyl chloride, polyvinylacetate, copolymers of vinyl acetate and vinyl chloride, polyamide,polycarbonate, polystyrene, etc., can be used. Examples of suitablematerials include Magna Nylon Supported Membrane from GE Osmonics(Minnetonka, Minn.), Novylon Nylon Membrane from CUNO Inc. (Meriden,Conn.) and Durapore Membrane from Millipore (Billerica, Mass.).

The matrix materials may be slit, cut, die-cut or punched into a varietyof shapes prior to incorporation into the immunoassay test slide 2 ofthe present invention.

Other porous materials suitable for the absorbent carrier 12 may includenatural, synthetic, or naturally occurring or synthetically modifiedmaterials: papers (fibrous) or membranes (microporous) of cellulosematerials such as paper, cellulose, and cellulose derivatives such ascellulose acetate and nitrocellulose, fiberglass, glass fiber, cloth,both naturally occurring (e.g., cotton) and synthetic (e.g., nylon);porous gels such as silica gel, agarose, dextran, and gelatin; porousfibrous matrices; starch based materials, cross-linked dextran chains;ceramic materials; olefin or thermoplastic materials including films ofpolyvinyl chloride, polyethylene, polyvinyl acetate, polyamide,polycarbonate, polystyrene, copolymers of vinyl acetate and vinylchloride and combinations of polyvinyl chloride-silica; and the like.This list is representative, and not meant to be limiting.

The porous materials, and specifications, for the fluid flow matrix setforth in U.S. Pat. No. 5,726,010, for example, mentioned previously maybe used in the immunoassay test slide 2 of the present invention, andsuch disclosures are incorporated herein by reference.

One or more analyte capture reagents are immobilized on the fluid flowmatrix 12 and situated thereon above the bottom opening 40 formed in thebottom piece 8 and beneath the top opening 38 formed in the cover piece6. The analyte capture reagent is a molecule which is bound to thematrix and which has a specific affinity for an analyte of interest.Preferably, the affinity arises by virtue of the reagent possessing acomplementary three-dimensional structure to the analyte, e.g., as seenin the relationship between an enzyme and a substrate or an antigen andan antibody. Within a given pair, either member may be considered to bethe analyte or the capture reagent. This definition serves only todifferentiate the component to be detected in the sample (i.e., theanalyte) from the reagent included in the immunoassay test slide 2(i.e., the analyte capture reagent).

As stated above, different analyte capture reagents may be immobilizedon the matrix 12 for different tests. For example, one analyte capturereagent may include an immobilized antibody specific for felineimmunodeficiency virus, and another may include an immobilized antibodyspecific feline leukemia virus. A single biological sample (e.g., asample of feline serum) may be deposited on the slide 2 and assayed forthe presence of one or both viruses. The immobilized analyte bindingpartner may be pre-deposited on the immunoassay test slide 2 prior toits assembly, or may be deposited on the assembled slide and optionallyheat dried.

Example: T4 Immunoassay

A method of performing an assay using the immunoassay test slide 2 ofthe present invention will now be described, and reference should be hadto FIGS. 21A-28 of the drawings. In the example shown in FIGS. 21A and21B, an assay is performed of thyroxine (T4) using anantibody-horseradish peroxidase conjugate. For this description,reference should further be had to the aforementioned Rich, et al.published application and the description of the structure and operationof the chemical analyzer therein, but also to FIG. 22, which illustratesa chemical analyzer 64 similar to that disclosed in the Rich, et al.published application, and related FIGS. 23-28 showing referencedcomponents of the chemical analyzer. The assay consumables areenvisioned to include the T4 slide; a “CTdx” diluent drawer packagewhich includes a conjugate, a wash buffer and a substrate; and a sample,such as serum or plasma.

The first step in the method is to load all of the consumables into thechemical analyzer. More specifically, one or more of the immunoassaytest slides 2 of the present invention are loaded onto a slide insertermechanism 20 (see FIG. 23) (also reference no. 20 in the Rich, et al.application) situated behind doors 16 (see FIG. 23) of the chemicalanalyzer (also reference no. 16 in the Rich, et al. application), whichopen to gain access to one of two slide inserter mechanisms. The slideinserter mechanism loads the immunoassay test slide, among otherchemical reagent test slides, onto a slide transport mechanism 26 (seeFIG. 24) (also reference no. 26 in the Rich, et al. application). Theslide transport mechanism 26 selectively positions the immunoassay testslide under a fluid sample metering device 84 (see FIG. 24) (alsoreference no. 84 in the Rich, et al. application) and above either orboth of a reflectometer 684 (see FIG. 25) (also reference no. 684 in theRich, et al. application) and a fluorometer 654 (see FIG. 26) (alsoreference no. 654 in the Rich, et al. application) to conductcolorimetric or fluorescence measurements.

A predetermined volume of fluid sample, such as blood, serum or thelike, is added to a sample vial 242 (see FIG. 27) (also reference no.242 in the Rich, et al. application), and the sample vial is placed onthe slide inserter mechanism 20 in a well 206 (see FIG. 27) (alsoreference no. 206 in the Rich, et al. application) for holding the vial.Furthermore, separate vials containing a liquid conjugate reagent, awash reagent and a detector reagent (TMB) can be loaded into respectivewells formed in a diluent drawer 136 (see FIG. 28) behind door 132 (seereference nos. 36 and 32 in the Rich, et al. application) of thechemical analyzer or in any other suitable location so long as thereagents are accessible to the analyzer's fluid metering system (seeStep 1 in FIG. 21A).

Then, in Step 2 (see FIG. 21A), the fluid sample and the conjugatereagent are mixed by the metering device 84 of the chemical analyzer 64either in a conjugate vial or in a separate empty vial situated in thediluent drawer 136 behind door 132. The mixing of the sample andconjugate may be performed in the manner disclosed in the Rich, et al.published application. The sample/conjugate mixture is then incubatedwithin the chemical analyzer 64 at a predetermined temperature (forexample, 37° C.) for a predetermined period of time (for example, fiveminutes). When the sample and conjugate reagent are mixed, the T4disassociates from the serum binding proteins in the fluid sample andthen binds to the T4-antibody*HRP.

After incubation, a relatively small amount (preferably 8 microliters)of the sample/conjugate mixture is dispensed on the immunoassay testslide 2 by the metering device 84 of the chemical analyzer, as set forthin Step 3 shown in FIG. 21A. Here, unbound T4-antibody*HRP binds to theimmobilized capture reagent spot on the fluid flow matrix 12, which inthe example shown is T3-PAA. One useable method of aspirating thesample/conjugate mixture from the vial and depositing the mixture on theimmunoassay test slide 2 by the metering device 84 of the chemicalanalyzer is disclosed in the aforementioned Rich, et al. publishedapplication.

Now, in Step 4 shown in FIG. 21A, the immunoassay test slide 2 is washedmultiple times. More specifically, the test slide preferably undergoesfour washes using 8 microliters of wash reagent for each wash dispensedby the metering device 84 of the chemical analyzer, each wash beingpreferably spaced apart in time by about 30 seconds. The metering device84 of the chemical analyzer aspirates preferably at least 32 microlitersof wash reagent from a vial in the diluent drawer containing suchsolution, and periodically dispenses preferably 8 microliters of thewash reagent onto the slide 2. With such washes, the T4-antibody*HRPbound to serum T4 is washed away.

The wash solution is a liquid reagent that serves to remove unboundmaterial from at least the central portion of the fluid flow matrix 12situated above the bottom opening 40 in the bottom piece 8 or in theregion of the matrix 12 which is subjected to measurement tests by thereflectometer or fluorometer. The wash reagent contains a surface activeagent, such as a surfactant, or any other component capable of allowingthe wash to wet the fluid flow matrix 12. Some other examples of washreagents are alcohol (e.g. methanol) or any other water miscible organicsolvent. Thus, in the example shown in FIGS. 21A and 21B, unbound sampleand unbound antibody-horseradish peroxidase conjugate are displaced bythe wash reagent. There should be sufficient time allotted betweendispensing the sample/conjugate mixture on the slide (Step 3 in FIG.21A) and the start of the multiple wash step (Step 4 in FIG. 21A) tomaximize the binding of the sample analyte to the specific bindingreagent.

After waiting about 30 seconds after the fluid flow matrix 12 is washedthe fourth time, the substrate, or detector reagent, is dispensed on theimmunoassay test slide 2 in a predetermined volume, preferably about 8microliters (see Step 5 in FIG. 21A). The substrate, or detectorreagent, produces a detectable signal upon reaction with theenzyme-antibody conjugate at the central portion of the matrix 12. Anexample of a detector reagent, or substrate, which produces an insolubleend product following reaction with the enzyme, horseradish peroxidase,is tetramethybenzidine, or TMB, such as TMBlue, available from TSIIncorporated of Worcester, Mass., Part no. TM 101. The end productproduced by the TMBlue substrate is a dye that absorbs light. In theexample shown in FIGS. 21A and 21B, the T4-antibody*HRP bound to thespotted T3-PAA develops color, which is detectable by the reflectometer684 of the chemical analyzer. Alternatively, a detector reagent, orsubstrate, may be chosen to cause light to be emitted from the slideupon eradiation, e.g. fluorescence, by a fluorometer 654 of the chemicalanalyzer. The degree of color change of the matrix is reflective of theamount of analyte in the fluid sample. Examples and descriptions ofvarious conjugate reagents, specific binding reagents, fluid flowmatrices, wash reagents and detector reagents and substrates aredisclosed in the aforementioned U.S. Pat. No. 5,726,010 and the patentsand publications cited therein, and such disclosures are incorporatedherein by reference.

FIG. 21B shows a graph plotting exemplary test results of reflectancemeasurements versus time for a T4 slide following the steps of themethod described above and shown in FIG. 21A. Reflectance measurementsare preferably performed at 645 nanometers every 15 seconds on the T4slide spotted with sample (labeled as “7.0 ug/dL T4” in FIG. 21A) andpreferably a control T4 slide not spotted with sample (labeled as “0.0ug/dL T4” in FIG. 21A). Exemplary reflectance measurements of both thesample T4 slide (shown by the line with diamonds) and the control T4slide (shown by the line with squares) are plotted in the graph of FIG.21B.

Example: Feline Pancreatic Lipase (fPL) Immunoassay

The manufacture and use of a Feline Pancreatic Lipase (fPL) immunoassaytest slide formed in accordance with the present invention will now bedescribed. The fPL immunoassay test slide may have the structure shownin the embodiment of FIGS. 9-20 or the embodiments shown in FIGS. 1-4,as will be described in greater detail.

Preferably, in forming the fPL immunoassay test slide, the porous matrix12, which is preferably formed from a Fusion 5™ absorbent material, isplaced into the slide housing 4 having a crystal clear (i.e., lighttransmissive) bottom side. Ten microliters of fPL 17A reagent particlesare spotted onto the porous matrix (either on the top side or the bottomside of the matrix). The spotted slides are then dried in a dryingtunnel for about 0.5 hour at about 95° Fahrenheit. The dried slides maythen be used immediately or stored at preferably about 4° Celsius.

A two-step protocol for testing for Feline Pancreatic Lipase isdescribed below and shown in FIGS. 5 and 6 of the drawings. Sixteenmicroliters of sample are aspirated from a sample cup, and thendispensed onto the test slide at 4 microliter aliquot portions. Then, 16microliters of conjugate are aspirated from the conjugate cup, and thendispensed onto the slide at 4 microliter aliquot portions. After this,24 microliters of a wash buffer are aspirated from the wash cup, andthen dispensed onto the slide at 4 microliter aliquot portions. This isfollowed by 24 microliters of TMB substrate (i.e., a detector reagent)being aspirated from the TMB cup, and dispensed onto the slide at 4microliter aliquot portions. Finally, blue color development of the spotof the matrix is recorded by optical reflectances (at 645 nanometers)for 60 seconds in preferably in one second intervals. The data isplotted, and the resulting linear curve fitted in Excel™ to obtain thekinetic read of the assay. The fPL progress curve and the fPL calibrateddose curve for this two-step protocol using the immunoassay test slideof the present invention are respectively shown in FIGS. 5 and 6 of thedrawings.

A one-step protocol using the fPL immunoassay test slide of the presentinvention will now be described. In this one-step protocol, the sampleand conjugate are mixed in the chemical analyzer's pipette tipbeforehand and the mixture is then dispensed. An in-tip-mixing one-stepprotocol for testing a multiple of three fPL immunoassay slides isdescribed below.

Typically, 80 microliters of sample are first aspirated from the samplecup into the pipette tip of the chemical analyzer. 80 microliters ofconjugate are then aspirated from the conjugate cup into the analyzer'spipette tip containing the sample. The sample and conjugate are mixedinside the analyzer's pipette tip for 10 seconds with a tip-mix-volumeof 10 microliters.

For a multiple premix dispensing protocol, 30 microliters of thesample/conjugate premix are dispensed onto the immunoassay test slidesof the present invention at 10 microliter aliquot portions.

For a single premix dispensing protocol, 15 microliters of thesample/conjugate premix are dispensed onto the immunoassay test slidesof the present invention at 15 microliter aliquot portions preferablywith a 90 second post-premix dispense time delay. The post-premixdispense time delay is preferably provided to permit sufficientincubation time for the immuno-reaction on the slides.

Then, 30 microliters of a wash buffer are dispensed onto the slides at10 microliter aliquot portions. Following this, 12 microliters of theTMB substrate are dispensed onto the slides at 12 microliter aliquotportions. Finally, blue color development of the spot on the test slidematrix 12 is recorded by optical reflectance (OR) at 645 nanometers for60 seconds in preferably two second intervals. The data are then plottedto obtain an end point read of the assay. For the in-tip-mixing one-steptest protocol, the spec fPL calibrator dose curves corresponding to thesingle and multiple premix dispense are respectively shown in FIGS. 7and 8 of the drawings.

The color change of the slide 2 detected by the reflectometer, or thefluorescence of the slide detected by the fluorometer, of the chemicalanalyzer may be measured quantitatively or qualitatively to determinethe amount of analyte in the fluid sample (see FIG. 21B and Step 6 inFIG. 21A). It is envisioned that the immunoassay test slide 2 of thepresent invention may be loaded into the chemical analyzer with otherimmunoassay slides or with dry chemistry reagent test slides, the slidesbeing tested concurrently. Another advantage of the immunoassay testslide over other conventional methods and devices for performing assaysis the minute quantity of fluid sample and liquid reagents required fordetecting the presence of an analyte in the fluid sample. In oneconventional immunoassay test device commonly referred to by thetrademark SNAP and manufactured by IDEXX Laboratories, Inc.,approximately 1,330 microliters of sample and liquid reagents aretypically required to perform the assay and obtain detectable results.However, with the immunoassay test slide 2 of the present invention,less than 100 microliters of sample and liquid reagents are required toperform an assay to obtain detectable results.

The immunoassay test slide 2 of the present invention may be formed byplacing a die cut section of porous carrier matrix 12 from a sheet ofthe same material between a cover piece 6 and a bottom piece 8 of aplastic material, such as polystyrene, specifically shaped to bematable.

The two pieces may be joined together by applying heat or an adhesive todefine a substantially leakproof housing 4 in which resides the porouscarrier matrix 12. The porous carrier matrix 12 may be spotted with animmobilized specific binding reagent prior to its insertion between thetwo mating slide pieces, or may be spotted with the specific bindingreagent and heated to a specific temperature and for a predeterminedperiod time to dry and immobilize the binding reagent in the centralportion of the matrix 12. If a bottom opening 40, formed in the bottompiece 8 of the immunoassay test slide 2, is provided, then prior to theinsertion of the porous carrier matrix 12 between the cover piece 6 andthe bottom piece 8, a thin sheet of transparent (clear) material 42,such as Mylar, is preferably used and placed within the interior cavity10 defined by the slide housing 4 over the bottom opening 40.Alternatively, no such bottom opening or covering sheet is required ifthe bottom piece 8 of the slide is formed from a light transmissible ortransparent material, such as polystyrene.

Other embodiments of the immunoassay test slide 2 of the presentinvention are shown in FIGS. 1-4. Each of the test slides 2 of thesefurther embodiments includes a slide housing 4, a porous carrier matrixor membrane 12, such as described previously, and a film or cover sheet70. As shown in FIGS. 1-4, the slide housing 4 is preferably trapezoidalin overall shape, but may be rectangular or square. Thus, the slidehousing includes a front wall 44, a rear wall 46 situated opposite thefront wall, and two opposite lateral walls 48. If the immunoassay testslide housing 4 is rectangular in shape, than each wall 44-48 isperpendicularly joined to its next adjacent wall. If the immunoassaytest slide housing has a trapezoidal shape, such as shown in FIGS. 1-4,then the front and rear walls 44, 46 are generally parallel to eachother, and the rear wall 46 has a length which is greater than that ofthe front wall 44, and the opposite lateral walls 48 are non-parallel toeach other and mutually converge from the rear wall 46 toward the frontwall 44. The embodiment of the immunoassay test slide shown in FIG. 4 issimilar in structure to that of the immunoassay test slide shown inFIGS. 1-3 except that the two opposite lateral walls 48 are longer thanthose of the test slide shown in FIGS. 1-3, giving the embodiment of theslide housing shown in FIG. 4 an elongated trapezoidal shape. As withthe embodiments described previously, the immunoassay test slides 2shown in FIGS. 1-4 may include an indexing notch 50 for properorientation of the test slide on an analytical instrument, and lateralside recesses 54 used for loading the test slides on an analyticalinstrument, in the same manner and in the same locations as the notchand lateral side recesses included in the dry chemistry test slidesdisclosed in the aforementioned Heidt, et al. patent and the Rich, etal. published application.

In the embodiments of the immunoassay test slides of the presentinvention shown in FIGS. 1-4, the test slide housing 4 includes arecessed portion 72 of the top side 74 thereof to define a recess orcavity 10 which may be square, rectangular or even trapezoidal in shape.This cavity 10 is dimensioned to at least partially receive therein theporous carrier matrix 12. The matrix 12 performs the same function andmay be made from the same material as the matrix described previouslywith respect to the other embodiments of the immunoassay test slide,that is, for holding a specific binding reagent and for absorbing apredetermined volume of fluid sample and conjugate reagent. Furthermore,the porous carrier matrix 12 is formed in the same manner as describedpreviously with the other embodiments of the immunoassay test slide, andis preferably dimensioned to be slightly smaller than the dimensions ofthe cavity 10 formed in the slide housing 4 so that its lateral edges 76are spaced slightly away from the interior side walls 78 of the slidehousing 4 defining the cavity 10 so as to define a channel or well 62between the matrix 12 and the interior side walls 78 at least partiallyabout the periphery of the housing. As with the other embodiments, thischannel or well 62 is provided to receive any overflow of fluid sample,reagent or wash solution from the matrix 12 which is envisioned tobecome saturated with such fluids. The well or channel 62 providescapacity in excess of the volume of fluid sample, reagents and washsolutions saturating the porous matrix 12. The preferred volume of thecavity 10 defined by the recessed portion 72 of the housing 4 is thesame as that described previously with respect to the other embodimentsof the immunoassay test slides.

The preferred material from which the porous matrix 12 is formed isreferred to by the trademark Fusion 5™ available from Whatman, Inc.,USA, which is a glass fiber-based material that contains a plasticbinder.

Preferably, the slide housing 4 is formed from crystal polystyrene,although it may be formed from the same materials which were describedpreviously with respect to the other embodiments of the immunoassay testslide. The crystal polystyrene is transparent or at least translucent.However, it is envisioned that a bottom opening 40, such as shown inFIG. 10, may be formed through the bottom side of the housing 4 if thehousing is formed from a less light transmissive or opaque material.Then, a transparent or clear (light transmissive) thin sheet of material42 (see FIG. 11), such as a Mylar film, may be placed over the bottomopening and adhesively joined or heat sealed to the inner surface of thebottom side in alignment with the recess or cavity 10 of the slidehousing, that is, interposed between the inner surface of the bottomside of the housing and the absorbent porous material 12, to insure theleakproofness of the housing 4. The crystal styrene material from whichthe slide housing 4 is preferably formed allows visible or infraredlight, and more preferably, light at a wavelength of about 645nanometers, to permeate therethrough. Thus, light emitted by thereflectometer 684 or fluorometer 652 of the analytical instrument willpass through the transparent bottom side of the slide housing 4 when theanalytical instrument is conducting reflectance or fluorescencemeasurements on the immunoassay test slide.

Preferably, a recessed ledge 80, raised above the floor of the recessedportion 72 that receives the matrix 12 but slightly below the surface ofthe top side 74 of the slide housing, is formed on at least two oppositesides of the recess or cavity 10. Preferably, each ledge 80 includes anelongated rib 82 which protrudes slightly above and outwardly from thetop surface of the ledge 80. As will be seen, the elongated ribs 82 areused as “energy directors” and are provided for welding purposes. Theribs 82 bond to the flat underside surface of the covering film 70 toaffix the covering film 70 to the slide housing 4 thereby securing thematrix 12 within the recess 10 under the film covering when the slide isbeing assembled. Furthermore, the top side 74 of the slide housing mayinclude a bar code 86 situated thereon to identify the type of reagentused on the slide, which bar code 86 is read by an optical code readerforming part of the chemical analyzer.

A thin polystyrene film 70 is placed over the porous matrix 12 andpreferably resides in or above the recess or cavity 10 formed in the topside 74 of the slide housing 4. Preferably, this polystyrene film 70 hasa thickness of about 0.2 millimeters. The film 70 is preferablydimensioned to closely fit within the recess 10 in which the energydirectors or ribs 82 reside, the opposite interior side walls 78 of theslide housing 10 being situated and dimensioned to help position thecovering film 70 therebetween.

The polystyrene covering film 70 includes an opening 88 formed throughthe thickness thereof, in much the same way as the cover piece 6 of theimmunoassay test slides described previously has an opening 38. The topopening 88 is provided so that a precise amount of a sample fluid, suchas blood, serum and the like, and reagents may be metered onto the testslide 2 and deposited on the porous matrix 12 situated under the opening88, by a sample metering device 84 of the dry chemistry analyticalinstrument such as disclosed in the aforementioned Heidt et al. '229patent and the Rich, et al. published application. This top opening 88may be circular, as shown in FIGS. 1-3, rectangular, or oval orelongated in shape, as shown in FIG. 4. Preferably, the opening 88formed through the top film portion 70 has a width along a minor axisthereof of between about 6 millimeters and about 12 millimeters, andmore preferably about 10 millimeters, if the opening is oblong in shape,and has a diameter of between about 6 millimeters and about 12millimeters, and more preferably about 10 millimeters, if the opening iscircular in shape. The covering film 70 is situated on the test slidehousing 4 by closely positioning the covering film within the recess 10defined by the lateral walls 78 of the housing so that the film 70completely covers the porous matrix 12 situated within the recess orcavity 10 of the slide housing. The covering film 70 may be joined tothe slide housing 4 by an adhesive, or by sonic welding or heat stampingthe film to the housing. Even more preferably, the underside surface ofthe film 70 rests on the ledges 80 and contacts the energy directors orribs 82, and the film 70 is joined to the ledges 80 by sonic weldingusing the energy directors 82.

Although illustrative embodiments of the present invention have beendescribed herein with reference to the accompanying drawings, it is tobe understood that the invention is not limited to those preciseembodiments, and that various other changes and modifications may beeffected therein by one skilled in the art without departing from thescope or spirit of the invention.

What is claimed is:
 1. An immunoassay test slide, which comprises: ahousing, the housing defining a cavity; and a fluid flow matrix, thefluid flow matrix being disposed within the cavity of the housing forreceiving a fluid sample containing an analyte, or one or more liquidreagents; wherein the housing includes a bottom side which is formedfrom a light transmissive material, a top side, the top side including arecessed portion to at least partially define the cavity for receivingthe fluid flow matrix, a front side, a rear side situated opposite thefront side, and opposite lateral sides, the cavity being situatedbetween the front and rear sides and between the opposite lateral sidesof the housing; and wherein the test slide further comprises: a top filmportion, the top film portion being situated on the housing opposite thebottom side and in alignment with and covering the fluid flow matrix,the top film portion having an opening formed through the thicknessthereof which is in communication with the cavity of the housing andwhich is provided to receive the fluid sample containing an analyte orthe one or more liquid reagents, the fluid flow matrix being disposedwithin the housing and situated in alignment with the top opening formedin the film portion to receive thereon the fluid sample or the one ormore liquid reagents; and wherein the top side of the housing includesat least one recessed ledge, the at least one recessed ledge beingsituated adjacent to the recessed portion which receives the fluid flowmatrix, the at least one recessed ledge having a top surface on which aportion of the underside surface of the top film portion rests.
 2. Animmunoassay test slide as defined by claim 1, wherein the housing has agenerally trapezoidal shape, and wherein the front and rear sides of thehousing are generally parallel to each other and the rear side is longerthan the front side.
 3. An immunoassay test slide as defined by claim 1,wherein the top film portion is formed from a polystyrene material. 4.An immunoassay test slide as defined by claim 1, wherein the top filmportion has a thickness of about 0.2 millimeters.
 5. An immunoassay testslide as defined by claim 1, wherein the opening formed through the topfilm portion is one of circular or oblong.
 6. An immunoassay test slideas defined by claim 5, wherein the opening formed through the top filmportion has a width along a minor axis thereof of between about 6millimeters and about 12 millimeters if the opening is oblong in shape,and has a diameter of between about 6 millimeters and about 12millimeters if the opening is circular in shape.
 7. An immunoassay testslide as defined by claim 6, wherein the opening formed through the topfilm portion has a width along a minor axis thereof of about 10millimeters if the opening is oblong in shape, and has a diameter ofabout 10 millimeters if the opening is circular in shape.
 8. Animmunoassay test slide as defined by claim 1, wherein the slide housingis formed from a polystyrene material.
 9. An immunoassay test slide asdefined by claim 1, wherein the top side of the slide housing includes abar code situated thereon.
 10. An immunoassay test slide as defined byclaim 1, which further comprises: at least one immobilized analytecapture reagent situated on the fluid flow matrix in alignment with theopening in the top film portion.
 11. An immunoassay test slide asdefined by claim 1, wherein the housing includes a side wall surroundingthe cavity, at least a portion of the side wall being spaced laterallyfrom the fluid flow matrix to define therebetween a channel forreceiving an excess volume of the fluid sample or the one or more liquidreagents.
 12. An immunoassay test slide as defined by claim 1, whereinthe front side of the housing has formed therein an orientation notch.13. An immunoassay test slide as defined by claim 1, wherein each of theopposite lateral sides of the housing has formed therein a recess. 14.An immunoassay test slide as defined by claim 1, wherein the fluid flowmatrix is formed from a porous material selected from the groupconsisting of natural, synthetic, or naturally occurring orsynthetically modified materials; fibrous materials; membranes ofcellulose materials, including paper, cellulose, and cellulosederivatives, including cellulose acetate and nitrocellulose, fiberglass,glass fiber, cloth, both naturally occurring, including cotton, andsynthetic, including nylon; porous gels, including silica gel, agarose,dextran and gelatin; porous fibrous matrices; starch based materials;cross-linked dextran chains; ceramic materials; olefin and thermoplasticmaterials, including films of polyvinyl chloride, polyethylene,polyvinyl acetate, polyamide, polycarbonate, polystyrene, copolymers ofvinyl acetate and vinyl chloride and combinations of polyvinylchloride-silica.
 15. The immunoassay test slide of claim 1, wherein thefluid flow matrix is formed from an absorbent material which is selectedfrom the group of materials consisting of: sintered polyethylene beads;nitrocellulose; glass fibers; and paper.
 16. The immunoassay test slideof claim 1, wherein the fluid flow matrix is formed from an absorbentmaterial which is treated with a surfactant.
 17. The immunoassay testslide of claim 1, wherein the light transmissive material from which thebottom side of the housing is formed can be permeated by visible orinfrared light.
 18. The immunoassay test slide of claim 1, wherein thelight transmissive material from which the bottom side of the housing isformed can be permeated by light having a wavelength of 645 nanometers.19. The immunoassay device of claim 1, further comprising an antibody orantigen disposed on the fluid flow matrix.
 20. The immunoassay testslide of claim 1, wherein the fluid flow matrix has a volume thatoccupies at least about 50% of the cavity.
 21. The immunoassay testslide of claim 1, wherein the fluid flow matrix has a volume thatoccupies at least about 60% of the cavity.
 22. The immunoassay testslide of claim 1, wherein the fluid flow matrix has a volume thatoccupies at least about 70% of the cavity.
 23. The immunoassay testslide of claim 1, wherein the fluid flow matrix has a volume thatoccupies at least about 80% of the cavity.
 24. The immunoassay testslide of claim 1, wherein the fluid flow matrix has a volume thatoccupies at least about 90% of the cavity.